The Mcm2-7- interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing

Masako Izumi; Takeshi Mizuno; Ken-ichiro Yanagi; Kazuto Sugimura; Katsuzumi Okumura; Naoko Imamoto; Tomoko Abe; Fumio Hanaoka

doi:10.1074/jbc.M117.779371

The Mcm2-7- interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing

¹ Accelerator Applications Research Group, RIKEN, Japan;
² Celllular Dynamics Laboratory, RIKEN, Japan;
³ Graduate School of Bioresources, Mie University, Japan;
⁴ Department of Life Science, Gakushuin University, Japan

↵* Corresponding author; email: mizumi{at}riken.jp

Author contributions: M. I., T. M., and F. H. conceived and designed the study. M. I. conducted the majoritymost of the experiments and wrote the paper. T. M. and K. Y. performed GST-pulldown assays. K. S. and K. O. performed DNA combing assays. N. I. and T. A. provided the helpful advice. F. H. wrote the paper and coordinated the study. All authors reviewed the results and approved the final version of the manuscript.

Abstract

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that Mcm10's conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) alpha, and proliferating cell nuclear antigen, while the chromatin loading of Cdc45 and Pol epsilon was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol epsilon.